Figure 1.
Histological sections of four-year-old AS(+) and control fish.
(A) abnormally developed ovary section of AS(+) carp. (B) normally developed ovary section of control carp. The scale bar = 100 µm; n, nuclear; PO, primary oocyte; PVO, previtellogenic oocyte; AO, atretic oocyte.
Figure 2.
Scatterplot charts of the duplicate spot intensities obtained from cDNA microarray analyses.
Cy3 and Cy5 dyes were used to label cDNA probes of the hypothalamus (A), pituitary (B), and ovary (C) prepared from AS(+) and control carp. The X coordinate value is the gene expression level (intensity value) in the test with the transgenic-Cy3 cDNA probes. The Y coordinate value is the other test with the control-Cy5 cDNA probes. Each dot represents a duplicate clone, and the green and red dots represent the clones up-regulated in AS(+) and control carp, respectively. The black dots indicate the clones with few changes between them.
Table 1.
List of differentially expressed genes (FDR <0.01 and fold change ≥2) in AS(+) hypothalamus relative to control hypothalamus.
Table 2.
List of differentially expressed genes (FDR <0.01 and fold change ≥2) in AS(+) pituitary relative to control pituitary.
Table 3.
List of differentially expressed genes (FDR <0.01 and fold change ≥2) in AS(+) ovary relative to control ovary.
Figure 3.
Pie diagram of the differentially expressed genes between AS(+) and control carp.
The functional category was based on sequence homologies and Gene Ontology (GO) enrichment analyses. The percentages are shown under their categories. (A) Percentage of expressed sequence tags (ESTs) based on novel ESTs, ESTs significantly similar with known genes (E values ≤1e−5) and ESTs function unknown. (B) Function known ESTs were classified into ribosomal/mitochondrial genes, metabolism, inflammation and immune, oogenesis and ovulation, hormone, other molecular functions.
Figure 4.
Schematic diagram comparing the differentially expressed genes of the hypothalamus, pituitary, and ovary.
Cytochrome c oxidase subunit I, NADH dehydrogenase subunit I, hemoglobin subunit alpha, and an unknown factor homologous to a Homo sapiens mRNA differentially expressed in malignant melanoma were exhibited in all three libraries, flavoprotein, follicle stimulating hormone beta-subunit (FSHβ), pro-opiomelanocortin, growth hormone (GH), DEAD (Asp-Glu-Ala-Asp) box polypeptide, cytochrome c oxidase subunit II, and III, and four unknown factors were identified in the pituitary and ovary libraries. Melanin-concentrating hormone precursor (MCH) gene was exhibited in the hypothalamus and ovary libraries.
Figure 5.
Validation of the differentially expressed genes using real-time quantitative RT-PCR.
(A) Hemoglobin subunit alpha (hbα), NADH dehydrogenase (nd), arginine methyltransferase (prmt), melanin-concentrating hormone precursor (mch), salmon gonadotropin-releasing hormone (sGnRH) in hypothalamus. (B) Growth hormone (gh), DEAD box polypeptide (dead), similar to melanoma inhibitory activity protein (mia), follicle stimulating hormone beta-subunit (fshβ), pyruvate kinase M2 (pkm2), secretogranin III (sgIII) in pituitary. (C) Cystatin precursor (cst), C-type lectin (lec), B-cell translocation gene 4 (btg4), GABA neurotransmitter transporter 1 (gnt1), high-mobility group box 1 (hmgb1), basigin (bsg), ZP2 (zp2), follicle stimulating hormone beta-subunit (fshβ), 40S ribosomal protein S14 (rps14), S100 calcium binding protein A1 (s100a1), ZP3 (zp3), superoxide dismutase (sod), geminin (gmnn), pentraxin (ptx) in ovary. β-actin was amplified with the target gene as a positive control in each well.
Table 4.
Plasma LHβ level of AS(+) and control groups in 4-year-old female fish.